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Cited by 50

The DOI matches its Crossref record; 52 of the 52 references it cites were found in a registry or an index.

Gene networks activated by specific patterns of action potentials in dorsal root ganglia neurons

Lee, Philip R., Cohen, Jonathan E., Iacobas, Dumitru A., Iacobas, Sanda, Fields, R. Douglas·2017·Scientific Reports
DOI 10.1038/srep43765PMC PMC5335607
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Abstract

Gene regulatory networks underlie the long-term changes in cell specification, growth of synaptic connections, and adaptation that occur throughout neonatal and postnatal life. Here we show that the transcriptional response in neurons is exquisitely sensitive to the temporal nature of action potential firing patterns. Neurons were electrically stimulated with the same number of action potentials, but with different inter-burst intervals. We found that these subtle alterations in the timing of action potential firing differentially regulates hundreds of genes, across many functional categories, through the activation or repression of distinct transcriptional networks. Our results demonstrate that the transcriptional response in neurons to environmental stimuli, coded in the pattern of action potential firing, can be very sensitive to the temporal nature of action potential delivery rather than the intensity of stimulation or the total number of action potentials delivered. These data identify temporal kinetics of action potential firing as critical components regulating intracellular signalling pathways and gene expression in neurons to extracellular cues during early development and throughout life.

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Figures

Figure 1 Stimulation patterns used in this study. (A) Shown are two patterns of electrical stimulation used throughout this study. For both 18/1 and 90/5 stimulation patters, an equal number of electrical pulses were delivered by bipolar stimulating electrodes across a Campenot chamber. 18/1 and 90/5 refer to 18 electrical stimuli delivered every minute (10 Hz) or 90 electrical stimuli (10 Hz) delivered once every 5 min for the duration of stimulation, respectively. In (B and C), is a representative image of a Campenot chamber used for long-term culture of DRG neurons showing axonal outgrowth (C) in the central compartment. Platinum electrodes deliver biphasic stimulation to both cell body compartments.
Figure 1 Stimulation patterns used in this study. (A) Shown are two patterns of electrical stimulation used throughout this study. For both 18/1 and 90/5 stimulation patters, an equal number of electrical pulses were delivered by bipolar stimulating electrodes across a Campenot chamber. 18/1 and 90/5 refer to 18 electrical stimuli delivered every minute (10 Hz) or 90 electrical stimuli (10 Hz) delivered once every 5 min for the duration of stimulation, respectively. In (B and C), is a representative image of a Campenot chamber used for long-term culture of DRG neurons showing axonal outgrowth (C) in the central compartment. Platinum electrodes deliver biphasic stimulation to both cell body compartments.CC BY · PubMed Central
Figure 2 Action potential pattern dependent regulation of gene-regulatory networks. Neurons were electrically stimulated with two patterns, 18/1 or 90/5 for 2 hr and 5 hr. (A) Hierarchical clustering of log-normalized microarray data showing differential expression of many hundreds of genes in electrically stimulated DRG neurons. (B) Frequency distribution of up- (red) and down-regulated (blue) genes in DRG neurons. Each gene is represented along the x-axis and corresponding log-normalized expression value. Numbers of up- and down-regulated genes are unbalanced, favoring greater transcriptional activation at 5 hr for 18/1 stimulus pattern compared to greater initial activation at the 90/5 stimulus pattern at 2 hr and down-regulation by 5 hr. (C) Venn diagram showing numbers of up- and down-regulated genes at 2 hr and 5 hr for both patterns of electrical activation. Total numbers of regulated genes are given in parenthesis for each time, pattern, and direction of change. In total, 2,501 genes were significantly upregulated by electrical stimulation and 3,424 were significantly down-regulated by electrical stimulation. Of these, a small subset of genes was co-regulated by both patterns. Only 9 activity-regulated genes were co-upregulated independently of pattern or stimulus duration in contrast to 22 genes suppressed by patterned activity. N = 4 replicate experiments per condition.
Figure 2 Action potential pattern dependent regulation of gene-regulatory networks. Neurons were electrically stimulated with two patterns, 18/1 or 90/5 for 2 hr and 5 hr. (A) Hierarchical clustering of log-normalized microarray data showing differential expression of many hundreds of genes in electrically stimulated DRG neurons. (B) Frequency distribution of up- (red) and down-regulated (blue) genes in DRG neurons. Each gene is represented along the x-axis and corresponding log-normalized expression value. Numbers of up- and down-regulated genes are unbalanced, favoring greater transcriptional activation at 5 hr for 18/1 stimulus pattern compared to greater initial activation at the 90/5 stimulus pattern at 2 hr and down-regulation by 5 hr. (C) Venn diagram showing numbers of up- and down-regulated genes at 2 hr and 5 hr for both patterns of electrical activation. Total numbers of regulated genes are given in parenthesis for each time, pattern, and direction of change. In total, 2,501 genes were significantly upregulated by electrical stimulation and 3,424 were significantly down-regulated by electrical stimulation. Of these, a small subset of genes was co-regulated by both patterns. Only 9 activity-regulated genes were co-upregulated independently of pattern or stimulus duration in contrast to 22 genes suppressed by patterned activity. N = 4 replicate experiments per condition.CC BY · PubMed Central
Figure 3 Opposing regulation of nerve growth factor and Rac signalling in DRG neurons. Pathway analysis of enriched canonical pathways identifies opposing regulation of Rac signalling by 18/1 and 90/5 patterns of stimulation. Rac signalling pathways were oriented to depict extracellular, cytoplasmic, and nuclear sites of action. Nodes in the pathway are up- (25, red) or down-regulated (11, green) depending on the regulation of target pathway genes. Shaded targets reflect multiple regulated components (e.g. MAPK pathway in 18/1 stimulation pattern). Multiple downstream pathways interacting through Rac, e.g., actin polymerization, translation and membrane ruffling, affect neurite outgrowth and dynamics.
Figure 3 Opposing regulation of nerve growth factor and Rac signalling in DRG neurons. Pathway analysis of enriched canonical pathways identifies opposing regulation of Rac signalling by 18/1 and 90/5 patterns of stimulation. Rac signalling pathways were oriented to depict extracellular, cytoplasmic, and nuclear sites of action. Nodes in the pathway are up- (25, red) or down-regulated (11, green) depending on the regulation of target pathway genes. Shaded targets reflect multiple regulated components (e.g. MAPK pathway in 18/1 stimulation pattern). Multiple downstream pathways interacting through Rac, e.g., actin polymerization, translation and membrane ruffling, affect neurite outgrowth and dynamics.CC BY · PubMed Central
Figure 4 Enrichment of activity-regulated transcription factor binding sites. Hierarchical clustering of 311 TFBS by site and condition. Cis-regulatory elements identified through evolutionary conserved regions (ECRs), promoters, and 5′-UTRs are clustered by % occurrence within a gene set and by importance factor (TF abundance X TF weighting) for 18/1 and 90/5 stimulation patterns at 2 hr and 5 hr. Background gene sets (305 genes) were determined by filtering microarray data to identify genes not regulated by either pattern at 2 hr and 5 hr (fold-change < |1.4|), and used to calculate weighting for TF analysis. Enriched TFBS are annotated for both % abundance and importance.
Figure 4 Enrichment of activity-regulated transcription factor binding sites. Hierarchical clustering of 311 TFBS by site and condition. Cis-regulatory elements identified through evolutionary conserved regions (ECRs), promoters, and 5′-UTRs are clustered by % occurrence within a gene set and by importance factor (TF abundance X TF weighting) for 18/1 and 90/5 stimulation patterns at 2 hr and 5 hr. Background gene sets (305 genes) were determined by filtering microarray data to identify genes not regulated by either pattern at 2 hr and 5 hr (fold-change < |1.4|), and used to calculate weighting for TF analysis. Enriched TFBS are annotated for both % abundance and importance.CC BY · PubMed Central
Figure 5 Top 10 candidate transcription factor binding sites enriched by stimulus pattern and duration. Top 10 candidate cis-regulatory elements are shown for genes activated by the 18/1 (A) and 90/5 (B) stimulation patterns after 2 hr and 5 hr. Importance factor scores >0.2 may be functionally significant as measured by enriched datasets for NF-κB and CREB binding site and downstream regulators of MAPK signalling in DRG neurons (Figure S2). Several candidate sites have high importance factor scores for multiple conditions, e.g. Tef. The TFBS used for this analysis are not differentiated by enhancer, repressor, or activation.
Figure 5 Top 10 candidate transcription factor binding sites enriched by stimulus pattern and duration. Top 10 candidate cis-regulatory elements are shown for genes activated by the 18/1 (A) and 90/5 (B) stimulation patterns after 2 hr and 5 hr. Importance factor scores >0.2 may be functionally significant as measured by enriched datasets for NF-κB and CREB binding site and downstream regulators of MAPK signalling in DRG neurons (Figure S2). Several candidate sites have high importance factor scores for multiple conditions, e.g. Tef. The TFBS used for this analysis are not differentiated by enhancer, repressor, or activation.CC BY · PubMed Central
Figure 6 NF-κB target gene PIM1 is up-regulated by patterned action potential activity. Protein expression levels of PIM1 following 90/5 stimulation for 5 hr were measured by immunoblotting. 10 μM MG132, a general proteasome and NF-κB inhibitor, was pre-incubated for 2 hr prior to stimulation. MG132 treatment reduced basal levels of PIM1 expression. GAPDH was used as a loading control.
Figure 6 NF-κB target gene PIM1 is up-regulated by patterned action potential activity. Protein expression levels of PIM1 following 90/5 stimulation for 5 hr were measured by immunoblotting. 10 μM MG132, a general proteasome and NF-κB inhibitor, was pre-incubated for 2 hr prior to stimulation. MG132 treatment reduced basal levels of PIM1 expression. GAPDH was used as a loading control.CC BY · PubMed Central

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Metadata

Authors
Lee, P. R., Cohen, J. E., Iacobas, D. A., Iacobas, S., & Fields, R. D.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-03-03
Volume
7
Issue
1
ISSN
2045-2322
DOI
10.1038/srep43765
PMC
PMC5335607
Cited by
50
References
52
Language
en
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